Cytotoxic T cells (CTL) are one of the cornerstones of the immune system. There is therefore much interest in understanding the mechanism by which they lyse targets, which is the overall goal of this proposal. Although significant strides have been made in our understanding of CTL lysis many questions remain open. Adsorption of CTL to targets via receptors triggers polarization of cytoskeletal elements in the CTL. This leads to reorientation of the Golgi apparatus (GA) towards the target binding site and secretion of channel forming proteins -perforins (PF) - which are thought to cause target cell lysis. Recent evidence suggest s that this sequence of events established with in vitro CTL clones does not operate in vivo induced CTL. Evidence is also accumulating that target lysis by channel formation may not be the universal mechanism of CTL lysis and that the target itself plays an active role in its lysis. We propose to explore several aspects of the concurrent models for CTL lysis in in vivo induced CTL, because they have not yet acquired abnormal properties in vitro. CTL will be highly purified and tested whether they show GA reorientation when bound to targets because this reaction is thought to signal proper target recognition. Next CTL will be tested as to their ability to insert PF channels into erythrocyte membranes. To induce this reaction we will use bifunctional anti-target anti-T cell receptor antibody conjugates. If CTL fail to insert channels into erythrocytes we will test whether removal of nuclei from targets inhibits target lysis. In drug inhibition studies we will examine whether continued lytic activity of the CTL on one hand and lytic susceptibility of the target on the other require protein synthesis prior to CTL-target binding. Preliminary data support the view that targets participate in CTL induced lysis. To explore how targets participate in lysis we propose several approaches, one of which is based on the ability of protein synthesis inhibitors to interfere with CTL lysis. Another is based on the concomitant occurrence of resistance to CTL lysis and resistance to glucocorticoids in one cell line. We propose to examine whether there indeed exists a correlation between glucocorticoid resistance and CTL sensitivity. This would provide support for a common pathway for both mechanisms of cell lysis. We also propose to select CTL resistant targets to explore whether they acquire resistance to PF and/or glucocorticoids. These variants will provide the tool to study the postulated suicide pathway. Finally, we will explore whether in vivo induced CTL change their mode of lysis upon prolonged culture in vitro and activate pathways of cell mediated lysis that have been described for cloned CTL.